16S-Seq of Streptococcus mitis: ivermectin cell culture
To extract bacterial DNA from liquid cultures, we used the DNeasy PowerSoil Pro kit (QIAGEN, 47016) and followed the manufacturers standard protocol included in the kit using 100l of the bacterial culture. Final DNA concentrations were determined using a QuBitTM 4 fluorometer (Thermo Fisher Scientific, Q33238) with the 1x dsDNA HS Assay kit (Thermo Fisher Scientific, Q33231). Eluates were stored at -20C until further use. Bacterial 16S rRNA sequencing was performed on the MinION Mk1C platform (Oxford Nanopore Technologies, United Kingdom of Great Britain). All samples were processed according to the manufacturers instructions provided with the 1-24 16S barcoding kit (Oxford Nanopore Technologies, SQK-16S024). In brief, the DNA concentration of all samples was measured using a QuBitTM 4 fluorometer (Thermo Fisher Scientific, Q33238) and the 1x dsDNA HS Assay kit (Thermo Fisher Scientific, Q33231). Samples were diluted using UltraPure DNase/RNase-free distilled water (Thermo Fisher Scientific, 10977-035) to reach a final input of 25ng DNA. Next, the samples were barcoded and amplified on a CFX Opus 384 Real-Time PCR system (Biorad, Cressier, Switzerland). Subsequently, the barcoded amplicons were cleaned up in multiple steps using the AMPure XP reagent (Beckman Coulter, A63881) in combination with the DynaMagTM-2 magnetic tube stand (Thermo Fisher Scientific, 12321D) and a HulaMixerTM (Thermo Fisher Scientific, 15920D). Finally, the sequencing libraries were pooled and sequenced using a flongle flow cell (Oxford Nanopore Technologies, FLG-001). Sequencing was performed for approximately 24h.